Nk3 receptor antagonist compound (nk3ra) for use in a method for the treatment of polycystic ovary syndrome (pcos)

ABSTRACT

A method for treating polycystic ovarian syndrome and related conditions with a compound (I): or a pharmaceutically acceptable salt thereof.

FIELD OF THE INVENTION

This invention is directed to the treatment of polycystic ovariansyndrome (PCOS) and related conditions.

BACKGROUND OF THE INVENTION

Polycystic ovarian syndrome (PCOS) is a condition that afflicts certainwomen. Hormonally, the disease may be characterized by an elevation inserum androgens. This elevation is thought to be the proximate cause forthe PCOS phenotype of hyperandrogenism (acne and hirsutism) whichtypifies these patients, as well as possibly contributing to features ofcentral adiposity and insulin resistance. The elevation of serumandrogens, along with the hallmark phenotype of anovulation andinfertility, has been traced to an elevation in serum luteinizinghormone (LH) levels, increased LH pulse frequency, and/or increasedserum LH/follicle stimulating hormone (FSH) ratio (See Hall J E, JEndocrinol Invest. 1998 October; 21(9):602-11).

At the time of filing there are no approved treatments for PCOS.“Off-label” therapies currently prescribed are aimed at eliminating thesymptoms of androgen excess. First-line treatment of PCOS is usually theoral contraceptive pill (OCP) for women in whom fertility is notimmediately desired. However, approximately half of these patients failto achieve adequate control of their androgenic symptoms with an OCP andrequire add-on anti-androgen therapy. Anti-androgen therapy is mostcommonly delivered as high-dose spironolactone, which carries the riskof potentially harmful electrolyte derangement. GnRH analogues are usedas the next line of therapy to reduce androgen levels, but given theirmarked potency they often induce chemical menopause, and thereforerequire add-back hormonal therapy. In addition to treatment with atherapy to reduce androgen levels, metformin may be administered as itis believed to improve menstrual regularity and fertility (as well asinsulin resistance). However, data on its efficacy in these endpoints isinconclusive. For women trying to conceive, additional treatment withclomiphene (+/−metformin) improves conception rates, but with theassociated risks of ovarian hyperstimulation and multiple pregnancies.

Therefore, in light of current “off-label” symptom-driven therapy withlimited efficacy and notable side effects, there is a need for anapproach that specifically targets the underlyinghypothalamic-pituitary-gonadal (HPG) pathophysiology of PCOS andmodulates LH pulsatility, with the goal of affording diseasemodification and concomitant symptom relief without significant adverseeffects.

DESCRIPTION OF THE INVENTION

Accordingly, we believe therapies that target the underlying HPGpathophysiology of PCOS and modulate LH pulsatility may be useful intreating PCOS. Furthermore, we believe that the NK3 receptor antagonist,3-(methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide(NK3RA):

and pharmaceutically-acceptable salts thereof, are useful in themodulation of LH pulsatility. One aspect of the invention is NK3RA andpharmaceutically-acceptable salts thereof for use in the modulation ofLH pulsatility. Another aspect of the invention is NK3RA andpharmaceutically-acceptable salts thereof for use in the treatment ofconditions in which modulation of LH pulsatility is beneficial. Afurther aspect of the invention is NK3RA and pharmaceutically-acceptablesalts thereof for use in the treatment of PCOS.

A further aspect of the invention is NK3RA for use in the modulation ofLH pulsatility. Another aspect of the invention is NK3RA for use in thetreatment of conditions in which modulation of LH pulsatility isbeneficial. A further aspect of the invention is NK3RA for use in thetreatment of PCOS.

Furthermore, current therapies that only treat symptoms derived fromelevated androgen levels result in some of the symptoms of PCOSremaining untreated. We believe, that NK3RA andpharmaceutically-acceptable salts thereof target the fundamentalpathophysiology of the disease and may have the potential to treatsymptoms not immediately derived from elevated androgen levels. Examplesof these symptoms include amenorrhea, oligomenorrhea and anovulation.NK3RA pharmaceutically-acceptable salt thereof may also be useful inrestoring fertility.

Accordingly another aspect of the invention relates to the use of NK3RAor a pharmaceutically-acceptable salt thereof in the treatment ofamenorrhea in PCOS. Another aspect of the invention relates to the useof NK3RA or a pharmaceutically-acceptable salt thereof in the treatmentof oligomenorrhea in PCOS. Yet another aspect of the invention relatesto the use of NK3RA or a pharmaceutically-acceptable salt thereof in thetreatment of anovulation in PCOS. Yet another aspect of the inventionrelates to the use of NK3RA or a pharmaceutically-acceptable saltthereof for the restoration of fertility.

Another aspect of the invention relates to the use of NK3RA or apharmaceutically-acceptable salt thereof in the manufacture of amedicament for modulating LH pulsatility. A further aspect of theinvention relates to the use of NK3RA or a pharmaceutically-acceptablesalt thereof in the manufacture of a medicament for the treatment ofPCOS.

A further aspect of the invention relates to a method of modulating LHpulsatility in a patient in need thereof, comprising administering tosaid patient a therapeutically effective amount of NK3RA or apharmaceutically acceptable salt thereof.

Another aspect of the invention relates to a method of treating PCOS ina patient in need thereof, comprising administering to said patient atherapeutically effective amount of NK3RA or a pharmaceuticallyacceptable salt thereof.

Another aspect of the invention relates to the use of NK3RA in themanufacture of a medicament for modulating LH pulsatility. A furtheraspect of the invention relates to the use of NK3RA in the manufactureof a medicament for the treatment of PCOS.

A further aspect of the invention relates to a method of modulating LHpulsatility in a patient in need thereof, comprising administering tosaid patient a therapeutically effective amount of NK3RA.

Another aspect of the invention relates to a method of treating PCOS ina patient in need thereof, comprising administering to said patient atherapeutically effective amount of NK3RA.

Accordingly another aspect of the invention relates to the use of NK3RAin the treatment of amenorrhea in PCOS. Another aspect of the inventionrelates to the use of NK3RA in the treatment of oligomenorrhea in PCOS.Yet another aspect of the invention relates to the use of NK3RA in thetreatment of anovulation in PCOS.

A further aspect of the invention is NK3RA andpharmaceutically-acceptable salts thereof for use in loweringtestosterone in a woman suffering from PCOS. A further aspect of theinvention is NK3RA for use in lowering testosterone in a woman sufferingfrom PCOS.

A further aspect of the invention relates to the use of NK3RA or apharmaceutically-acceptable salt thereof in the manufacture of amedicament for lowering testosterone levels in a woman suffering fromPCOS. A further aspect of the invention relates to the use of NK3RA or apharmaceutically-acceptable salt thereof in the manufacture of amedicament for lowering testosterone levels in a woman suffering fromPCOS.

Another aspect of the invention relates to a method for loweringtestosterone in a woman suffering from PCOS, comprising administering tosaid patient a therapeutically effective amount of NK3RA or apharmaceutically acceptable salt thereof. Yet another aspect of theinvention relates to a method of lowering testosterone in a womansuffering from PCOS, comprising administering to said patient atherapeutically effective amount of NK3RA.

A method of restoring fertility in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount ofNK3RA or a pharmaceutically acceptable salt thereof.

A method of treating PCOS comprising the step of determining whether thetestosterone level of a biological sample taken from a patient is higherthan the normal level and, if it is, treating said patient with atherapeutically effective amount of NK3RA, or a pharmaceuticallyacceptable salt thereof.

NK3RA or a pharmaceutically acceptable salt thereof may be of use in theprevention of any of the conditions mentioned hereinabove.

NK3RA or pharmaceutically acceptable salts thereof may also be of use intreating the following: precocious puberty, endometriosis, heavymenstrual bleeding, uterine fibroids, pre-eclampsia, androgenic acne,benign prostatic hyperplasia and/or androgenic alopecia.

Examples of pharmaceutically-acceptable salts are known in the art. Inone aspect, the pharmaceutically-acceptable salt of NK3RA is an acidaddition salt. In another aspect, NK3RA may be used as the free base.

The synthesis of 3-(methansulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide is described in patentapplication publication WO 2007/069977, the disclosure of which isincorporated herein in its entirety by reference. It may also beprepares according to example 1.

The NK3RA may be administered orally, parenteral, buccal, vaginal,rectal, inhalation, insufflation, sublingually, intramuscularly,subcutaneously, topically, intranasally, intraperitoneally,intrathoracically, intravenously, epidurally, intrathecally,intracerebroventricularly and by injection into the joints.

Preferably, administration will be orally by ingestion.

The quantity of the NK3RA to be administered will vary for the patientbeing treated and will vary from about 100 ng/kg of body weight to 100mg/kg of body weight per day. However, dosages can be readilyascertained by those skilled in the art from this disclosure and theknowledge in the art. Thus, the skilled artisan can readily determinethe amount of compound and optional additives, vehicles, and/or carrierin compositions and to be administered in methods of the invention.

Preferably the quantity of the NK3RA to be administered will vary forthe patient being treated and will vary from about 5 mg to 100 mg perday.

In another aspect, the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount ofthe NK3RA according to the present invention, or a pharmaceuticallyacceptable salt thereof, in association with at least onepharmaceutically acceptable excipient, carrier or diluent.

In another aspect the invention relates to a pharmaceutical compositioncomprising: the compound NK3RA;

mannitol and microcrystalline cellulose;croscarmellose sodium,hydroxypropyl cellulose,sodium lauryl sulphate, andmagnesium stearate.

In one aspect, the dose of NK3RA according to the present invention, ora pharmaceutically acceptable salt thereof, may be administered once aday. Population pharmacokinetic and pharmacodynamic analysisdemonstrates that NK3RA administered twice a day is better than once aday to maximally suppress testosterone during the entire dosinginterval. In another aspect, the dose of NK3RA according to the presentinvention, or a pharmaceutically acceptable salt thereof, may beadministered twice a day. In one aspect there is a period of at least 2hours between the 2 doses taken in the same day. In another aspect thereis a period of at least 4 hours between the 2 doses taken in the sameday. In another aspect there is a period of at least 6 hours between the2 doses taken in the same day. In another aspect there is a period of atleast 8 hours between the 2 doses taken in the same day. In anotheraspect there is a period of at least 10 hours between the 2 doses takenin the same day. In one aspect the total daily dosage is in the range 20mg to 180 mg of NK3RA. In another aspect the total daily dosage is inthe range 40 mg to 80 mg of NK3RA. In another aspect the total dailydosage is in the range 70 mg to 90 mg of NK3RA. In another aspect thetotal daily dosage is about 80 mg of NK3RA. In another aspect the totaldaily dosage is about 80 mg of NK3RA administered as a 40 mg dose twicea day.

The treatment of PCOS defined herein may be applied as a mono therapy ormay involve, in addition to the NK3RA, conjoint treatment withconventional therapy of value in treating PCOS. Such conventionaltherapy may include one or more of the following therapies currentlyprescribed that are aimed at eliminating the symptoms of androgenexcess.

First-line treatment of PCOS is usually the oral contraceptive pill(OCP) for women in whom fertility is not immediately desired. However,approximately half of these patients fail to achieve adequate control oftheir androgenic symptoms with an OCP and require add-on anti-androgentherapy (commonly high-dose spironolactone). GnRH analogues are used asthe next line of therapy, but require add-back hormonal therapy forwomen to avoid menopausal symptoms and the associated risks of exuberantGnRH antagonism. Metformin may also be used, as it is believed by someto improve menstrual regularity and fertility (as well as insulinresistance), although data on its efficacy in these endpoints isinconclusive. For women trying to conceive, clomiphene (+/−metformin)improves conception rates but with the associated risks of ovarianhyperstimulation and multiple pregnancies.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment.

Additional conventional therapy may include one or more of the followingcategories of agents: (i) antidepressants, (ii) atypical antipsychotics,(iii) antipsychotics, (iv) anxiolytics, (v) anticonvulsants, (vi)currently used Alzheimer's therapies, (vii) Parkinson's therapies,(viii) migraine therapies, (ix) stroke therapies, (x) urinaryincontinence therapies, (xi) neuropathic pain therapies, (xii)nociceptive pain therapies, (xiii) insomnia therapies and (xiv) moodstabilizers. Known treatments for the foregoing therapies may beemployed in combination with the invention described herein.

Such combination products employ NK3R A within the dosage rangedescribed herein and the other pharmaceutically active compound orcompounds within approved dosage ranges and/or the dosage described inthe publication reference.

Both non-clinical and clinical data on NK3RA have been obtained when itwas being developed for use in the treatment of schizophrenia and someof which are described herein. Also see, Expert Opin. Ther. Patents(2011) 21(5):637-655 and Simpson T R, Gadient R, Li Y, et al. Discoveryof AZD2624: a potent and selective NK3 antagonist to test the NK3hypothesis in schizophrenia Abstracts of Papers, 239th ACS NationalMeeting; 21-25 Mar. 2010: MEDI-35; San Francisco, Calif., USA

Non-Clinical Pharmacology of NK3 RA:

The activity of a compound in antagonising the hrNK3 receptor expressedin CHO cells may be measured in the following Intracellular CalciumMobilisation Assay:

CHO-K1 cells stably transfected with human recombinant Neurokinin-3receptor (hrNK3 CHO-K1) were cultured in T225 cm³ tissue culture flasksas monolayers in complete Ham's F-12 media (supplemented with 10% (v/v)FBS, 2 mM L-glutamine and 50 mg/ml Hygromycin B). Cultures aremaintained under standard tissue culture conditions. For experiments,PBS was used to wash cells free of culture media and Trypsin was used todetach cells from the flask surface. Cells were counted, pelleted bycentrifugation (100 g for 5 min) and resuspended in cell plating medium(UltraCULTURE by BioWhittaker 12-725F containing 200 mM L-Glutamine).The cells were pipetted into black walled, 384-well plate (clear bottom,poly-d-lysine coated plate (Biocoat, Becton Dickinson) at 10,000 cellsper well and maintained under standard tissue culture conditionsovernight.

Changes in intracellular free calcium were measured fluorometrically byloading hrNK3 CHO-K1 in 384-well plates with the calcium sensitivefluorescent dye, Fluo-4AM as described previously (Xiong et. al.,Bioorg. Med. Chem. Lett. 2001, 21, 1896). Briefly, cells were loaded for1 hour at 37° C. in assay buffer (Hanks' Balanced Salt Solutioncontaining 15 mM HEPES and 2.5 mM Probenecid) containing 4.4 M Fluo-4AM(dissolved in 10% (w/v) Pluronic F-127 in DMSO). Following dye loadingprocedure, assay buffer containing Fluo-4 AM was removed and replacedwith assay buffer alone.

A test compound was pre-dissolved in DMSO and incubated with cells to afinal concentration of 0.1% (v/v) DMSO for 15 minutes. Assays wereinitiated by the addition of Senktide (purchased from Bachem) and thetransient increase in Fluo-4-fluorescence monitored using a FLIPR(Fluorometric Imaging Plate Reader, Molecular Devices, Sunnyvale,U.S.A.).

NK3RA was tested and found to be an antagonist of the hrNK3 receptorexpressed in CHO cells.

Xiong H., Kang J., Woods J M., McCauley J P., Koether G M., Albert J S.,Hinkley L., Li Y., Gadient R A., Simpson T R. Synthesis and SAR ofsulfoxide substituted carboxyquinolines as NK3 receptor antagonists.Bioorg. Med. Chem. Lett. 2001, 21, 1896-1899

NK3RA is a potent and specific antagonist of the NK3 receptor. In vitrotarget engagement shows an IC50 2.24±0.57 nM (n=12), and specificity forNK3 over the other tachykinin receptors, NK1 and NK2, greater than 1200and 800 fold respectively.

Safety pharmacology studies were carried out on NK3RA and included teststo investigate possible effects on the central nervous system (CNS),cardiovascular system, and respiratory system. In a neurobehavioral testbattery in mice, oral doses of The NK3RA (100, 300, and 600 mg/kg [218,653, and 1306 μmol/kg]) produced transient deficits in gait andspontaneous motor activity, with full recovery apparent within 24 hours.There were no significant findings in the modified rat Irwin test afteroral treatment with The NK3RA at 46, 460, and 2022 mg/kg.

The NK3RA was only weakly active at the hERG channel in vitro, withslight (25%) inhibition of the hERG tail current at 97 μM. The NK3RA waswithout effect on the monophasic action potential of the guinea pigheart with IV doses up to 46 mg/kg (100 μmol/kg). A dog study thatinvolved electrocardiographic (ECG) telemetry measurements showed thatNK3RA treatment caused transient dose-related reductions in heart rateup to 29% at the highest dose tested (2000 mg/kg orally [4352 μmol/kg]).No other ECG parameter, including QTc interval duration, was affected indogs, and there was no effect seen on blood pressure. The NK3RA had nocardiovascular effects in rats at 46 mg/kg orally (100 μmol/kg), thehighest dose tested.

The NK3RA did not affect any respiratory parameters in rats up to 2000mg/kg (4400 μmol/kg).

The NK3RA showed no activity in a rat cocaine drug discrimination assayat oral doses up to 46 mg/kg.

Pharmacokinetics and Product Metabolism in Animals:

The NK3RA was absorbed at a moderate rate in rats and more rapidlyabsorbed in dogs following oral administration. At low doses, theestimated Cl_(p) of The NK3RA was moderate in rat (8.3 ml/min/kg) andlow in the dog (1.2 ml/min/kg). Bioavailability (F) was ˜100% in bothspecies indicating extensive absorption in these species. The volume ofdistribution (V_(z)) was moderate (1.6 L·kg in rat and 0.81 L/kg in dog)and consistent with the physico-chemical properties of the compound. Theelimination half-life (T½) ranged from 3.2 h (rat) to 9.5 h (dog). Asecondary peak in plasma concentration was observed for some dogs aftersingle or multiple dosing, possibly due to delayed absorption orenterohepatic recycling. Exposure increased with increasing dose of TheNK3RA, although the increases were less than dose proportional. TheNK3RA exposure decreased after multiple dosing in rats, indicatingeither changes in absorption or induction of The NK3RA metabolism, orboth, after multiple dosing. Elimination of NK3RA in the dog was notcomplete by 72 hours; however, no accumulation or reduction of NK3RA wasobserved after a 28-day repeat dosing regimen. T_(1/2) values rangedfrom 5.6 hours to 14.3 hours at the end of dosing for dogs dosed at 1000mg/kg/day (2180 μmol/kg/day). There were no consistent sex differencesobserved in the exposure to NK3RA in these 2 species.

Following oral administration of The NK3RA in the rat, apharmacologically active so ketone metabolite appeared rapidly in plasmabut slowly reached maximum concentration levels in all animals. Moderateaccumulation of the metabolite was observed after multiple dosing withthe NK3RA in both rats and dogs. The area under the plasmaconcentration-time curve from time 0 to 24 hours (AUC(0-24))metabolite-to-parent ratios were relatively constant with the increasein dose within each day, but increased after multiple doses of theNK3RA, ranging from 0.731 to 1.97 in the rat and from 0.0880 to 0.196 inthe dog, indicating that the NK3RA was highly metabolized in the rat andmoderately in the dog. T_(1/2) values ranged from 8.51 hours to 13.8hours on Day 27 and Day 28 for dogs dosed with 1000 mg/kg/day (2180μmol/kg/day) of the NK3RA. There were no consistent sex differencesobserved in the exposure to the metabolite in these 2 species.

Toxicology in Animals:

The NK3RA was well tolerated following single dosing up to the limitdose of 2000 mg/kg in rats and dogs, and following multiple dosing forthree month up to 2000 mg/kg/day in rats and up to 1000 mg/kg/day indogs. In rats, the highest doses produced a few clinical observationsand increased liver weights with no histological correlate.

In dogs, the testis, epididymis, and prostate were target organs inmales. In female dogs, ovarian uterine weights were reduced in treatedanimals. Increased liver weights were seen at mid and high doses andhepatobiliary toxicity in one high dose male. Thyroid weights wereincreased in the high dose group females. No other target organs havebeen identified.

Changes in the reproductive organ in the general toxicity andreproductive toxicity studies are entirely consistent with perturbationof regulatory hormones in animals with normal hormone homeostaticcontrol.

Reproductive Organ Observations in Animals:

In the rat 3 month study relative testis weight was lower than controlsat 200 and 2000 mg/kg/day (dose-related) and absolute weight was lowerat 2000 mg/kg/day. Relative epididymis weight was lower at 2000mg/kg/day. There were no NK3RA-related histological changes.

In the dog, the testis, epididymis and prostate weights were targetorgans, and no NOEL was established because effects were seen at alldose levels investigated in the study. Following the recovery period,weights of the testes had only partially returned to control values,while epididymis and prostate were comparable to controls. Epididymidesand prostate glands had gained maturity, but aspermia was present in alldogs and testicular immaturity remained. Although the sexual maturity ofthe dogs at study start was not known, the findings are suggestive ofdelayed maturity at least in the 1 month study.

Plasma testosterone levels were shown to be very low within by 4 hoursfollowing dosing, virtually totally suppressed throughout the study, andwere returning to normal levels by 48 to 72 hours after a final dose.Treatment of dogs with an antagonist of NK-1, -2 and -3, produced asimilar effect (Losco et al Toxicol. Pathol. 2007; 35: 310-322). Reducedtestis, epididymis and prostate weights were seen, with atrophy of allthree organs with epididymal oligospermia or aspermia and epithelialapoptosis and vacuolation. Similar effects were not seen in the rat. Theauthors considered the observations in the dog to be the result ofNK-3-mediated suppression of gonadotropin releasing hormone orluteinizing hormone (LH) and not a direct effect on the organs, becausealthough a secondary effect of NK antagonism on the tissues could not beruled out, there were no species-specific differences in toxicokineticsthat could explain the dog-specific observations

The antagonist of NK-1, -2 and -3, also produced reduced uterine andovary weights and uterine and ovarian immaturity.

During the 3-month study with NK3RA reductions in ovarian and uterineweights and an increased incidence of anestrus were observed in femaledogs, with complete recovery seen following 3 months off-dose. In rats,there were no findings in the female reproductive system during therepeat-dose toxicology studies with NK3RA. Disturbed estrus cycles wereseen with no adverse effects on mating performance or fertility in therat female fertility and embryofetal development study.

In female rats the No Adverse Effect Dose Level for fetal minorabnormalities and variants was 1000 mg/kg/day.

In female rabbits, the no effect dose level for maternal toxicity was 25mg/kg/day. The no adverse effect dose level for fetal observations was50 mg/kg/day.

Pharmacokinetics, Metabolism and Pharmacodynamics in Humans:

Pharmacokinetic properties have been investigated in healthy malesubjects. The NK3RA was quickly absorbed following oral dosing. Theelimination half-life for the NK3RA was approximately 7 hours. Both thearea under the curve (AUC) and maximum plasma concentration (Cmax)appeared to be dose proportional for both the NK3RA and the activemetabolite, up to approximately 80 mg. Renal elimination of the NK3RA orits metabolite was negligible. Following multiple dose administration,pharmacokinetic steady state was achieved within 4 days. At steadystate, the exposure to the metabolite was approximately 66% of theparent circulated; the accumulation of the NK3RA in the plasma wasminimal following QD dosing and was greater following BID dosing. NK3RApharmacokinetics appeared to be time independent. Exposure to the NK3RAin patients with schizophrenia was similar to that seen in healthysubjects.

Food Effects and Formulation Development:

Bioavailability of tablet and liquid suspension formulations is similar.Based on limited observations, oral administration of the NK3RAsuspension or tablets with food (a high-fat meal) increased the rate ofabsorption (suspension: 25% increase in Cmax; Tablet: 75% increase inCmax).

Clinical—Endocrine Effects:

In healthy male volunteer subjects in both the SAD and MAD studies, theNK3RA demonstrated a dose dependent reduction in serum testosterone inboth SAD and MAD studies with 1, 5, 10, 15, 40 and 80 mg tested in theSAD studies and 5 mg QD, 10 mg QD, 30 mg QD, 40 QD, 15 mg QD, 15 mg BID,or 30 mg BID tested in the MAD studies for up to 6 days.

In a phase II PoP schizophrenia trial using 40 mg QD, reduction in serumtestosterone was also an observed effect.

Therefore we have consistently seen reductions in total testosteronelevels durable up to 28 days dosing in male volunteers and maleschizophrenia patients.

A specific example of a pharmaceutical preparation of NK3RA is asfollows:

The NK3RA may be formulated as a 20 mg white film-coated tablet and iscompressed from granules containing 6.67% w/w of the NK3AR. Theformulation may be comprised of a dual-filler combination of mannitoland microcrystalline cellulose, croscarmellose sodium as a disintegrant,hydroxypropyl cellulose as a binder, and sodium lauryl sulfate aswetting agent. Magnesium stearate may be used as a lubricant. The NK3RAis size reduced before incorporation into the granulation. Such a tabletformulation has been shown to have suitable compaction and dissolutionproperties for clinical study.

Clinical Information in Females with PCOS:

The pharmacodynamics, safety and pharmacokinetics of a compound of thepresent invention, NK3RA, is being further evaluated in a randomized,double-blind, placebo-controlled of NK3RA when given in multiple dosesto females with PCOS.

In this type of clinical trial three doses of 20 mg once daily, 20 mgtwice daily and 40 mg twice daily of test compound are being compared toplacebo, and studied in parallel group design. The objectives of thestudy are to evaluate the changes from baseline of free and totaltestosterone on day 7 and 28 of dosing, to assess the safety andtolerability of the test compound in the target population, to measurethe plasma exposure of the test compound and metabolite in the targetpopulation, and to assess the PK/PD relationship of the test compoundand LH AUC, LH MPP and free and total testosterone. Exploratory analyseswill focus on changes from baseline of LH, DHEA, FSH, oestrogen,progesterone, prolactin, thyroid stimulating hormone, Insulin-likeGrowth Factor 1 [IGF-1](as surrogate for GH) and HbA1c on days 7 and 28,as well as the impact of treatment on patient reported outcomes andHRQoL as measured by change from baseline at day 28.

The study population includes women with PCOS aged 18-45. The study willenrol 14 PCOS subjects per dose cohort with a total of N=56 for theentire study (2 drop outs per group assumed to give 12 evaluablesubjects per dose). Patients are being dosed in the is study over aduration of 28 days.

Early Screening Baseline Treatment Follow-up Discontinuation Visitnumber Pre-screening 1 2 3 4 5 6 7 8 Relative to baseline −42 to −1 −28to −1 −1 +7 ± 1 +14 ± 1 +21 ± 1 28 ± 1 +42 ± 3 Inclusion/exclusion X X Xcriteria ICF issued X Informed consent X Demography X Medical/surgical Xhistory HIV, Hep B, Hep C X assessment Test for alcohol and X drugs ofabuse Randomisation X Dispense study X medication (a) Dispense dosing Xdiary (b) Administer morning X X dose at site with time recording (c)Assessment of X X X X compliance Return of unused X medication Recordconcomitant X X X X X X X X Medication Record Adverse X X X X X X X XEvents (d) (Abbreviated) X X X X Physical exam Height X Weight X X X X XX BMI X Supine BP and HR (e) X X X X X X 12 lead ECG (f) X X X X X XUrinalysis X X X X Urine pregnancy test X X X X X X X X HRQoL - SF-36 XX X Symptoms X X X X X X questionnaire Haematology X X X X X X X XClinical chemistry X X X X X X X X Screening X X X Testosterone (totaland free) High frequency LH X X X samples Every 10 mins for 8 hours (g)Well defined X X X testosterone (total and free), FSH. Hourly for 8hours (h) Single sample X X Testosterone (total and free, LH, FSH)Monitoring sample (i) X X X X X Estradiol (E2), progesterone, prolactin,TSH, T4 (total and free) HbA1c, IGF-1 Fast for 2 hours prior X X to and1 hour post morning dose PK samples (j) X X X Pre dose 4-beta X Xhydroxyl cholesterol and 6-beta hydroxyl testosterone Consent and bloodX sample for genetic analysis All labs central lab (a) First dose to betaken as an outpatient on day 1 (b) For PK and PD measurements recorddosing times on the day before the patient attends the site. Sitesshould contact subject by telephone to remind them (c) Patient takesmorning dose only on day 28 (d) SAEs will be recorded from signing ofthe informed consent, non-serious AEs will be recorded from the firstadministration of investigational product (e) Measure BP and HR oncepatient has rested supine for 10 minutes (f) On intensive monitoringdays 7 and 28 take ECG 2 hours post dose (g) LH sampling will beinitiated at approximately the same time on each visit (±1 hour).Intensive sampling should begin by 9 am (h) First sample pre-dose,within 30 minutes of dose (i) Sample to be taken ±1 hour of same timeeach day for all visits (j) PK sample times: Day 7 and 28: pre dose, 15min, 30 min, 1 hour, 1.5, 2, 3, 4, 6, 8. Early discontinuation singlesample.

Inclusion Criteria

For inclusion in the study subjects should fulfil the followingcriteria:

-   -   1. Provision of informed consent prior to any study specific        procedures    -   2. female subjects between the ages of 18 to 45 years        (inclusive)    -   3. Women has a diagnosis of polycystic ovary disease as defined        by the following        -   polycystic ovaries (previously documented ultrasound from            records are acceptable)        -   At screening free testosterone >ULN.        -   At screening total testosterone <5 nmol/L    -   4. Amenorrhea or oligomenorrrhea (defined as <=6 menses per        year)    -   5. Body Mass Index (BMI) between 18 and to 40 kg/m2 (inclusive)    -   6. Patient is permanently or surgically sterilized or who agrees        to maintain abstinence for the duration of study participation,        or who agrees to use/have their partner use effective methods of        birth control for the duration of their study participation        Permanent sterilisation includes bilateral salpingectomy but        excludes bilateral tubal occlusion. Effective Methods of birth        control within the study treatment period is defined as partner        use of male condom plus one of: spermicide, vasectomy, tubal        occlusion or an intrauterine device that does not contain        steroid hormones.

Exclusion Criteria

Subjects should not enter the study if any of the following exclusioncriteria are fulfilled:

-   -   1. Is peri-menopausal or has reached natural menopause—defined        as FSH>10 IU/L    -   2. Has menstruated within the last month    -   3. Clinically relevant disease and abnormalities (past or        present) which in the opinion of the investigator, may either        put the subject at risk to participate in this study or may        influence the results of the study or the subject's ability to        participate in the study    -   4. Significant illness, as judged by the investigator, within 2        weeks of Day−1    -   5. Patient has clinical, laboratory, or ECG evidence of        uncontrolled hypertension (defined as SBP of ≧160 mm Hg and/or        DBP of ≧□100 mm Hg), uncontrolled diabetes, HIV disease, or        significant pulmonary, renal, hepatic, endocrine, or other        systemic disease in the opinion of the investigator    -   6. Subjects who have had a hysterectomy or bilateral        oophorectomy or both; If the subject has had prior ovarian        cystcctomy(ies), unilateral oophorectomy, uterine surgery such        as myomectomy(ies) or polypoctomy(ies), etc., then these        subjects may be considered on a case-by-case basis by the        project Medical Advisor, with the aim to exclude anyone who no        longer has ovarian function or a functional endometrium.    -   7. Patient has a history of Gilbert's syndrome, infectious        hepatitis, or other significant hepatic disease (e. g. chronic        hepatitis, cirrhosis, autoimmune hepatitis, primary sclerosing        cholangitis, non-alcoholic steatohepatitis, or hereditary liver        disease) in the opinion of the investigator.    -   8. Patient has a history of gastric or small intestinal surgery        (including gastric bypass surgery or banding), or has a disease        that causes malabsorption.    -   9. Clinically significant abnormal ECG and/or abnormalities in        ECG as judged by the investigator at screening    -   10. A marked prolongation of QT/QTc interval (e.g., repeated        demonstration of a QTc interval >450 milliseconds (ms))    -   11. A history of additional risk factors for TdP (e.g., heart        failure, hypokalemia, family history of Long QT Syndrome)    -   12. The use of concomitant medications that prolong the QT/QTc        interval    -   13. Positive human immune deficiency virus (HIV), Hepatitis B or        Hepatitis C serology evaluations at the screening visit    -   14. Patient has a history of hypersensitivity to more than two        chemical classes of drugs, including prescription and        over-the-counter medications    -   15. Past (within 1 year of enrolment) or present alcohol or        substance abuse or a positive test for alcohol or drugs of abuse        or is a “recreational user” of illicit drugs or prescription        medications    -   16. Positive test for drugs of abuse or alcohol at the screening        visit    -   17. Patient consumes 3 or more alcoholic drinks per day. Note: 1        drink=12 oz. can/bottle of beer or 4 oz. of wine, or 1 oz. of        liquor    -   18. Enrolment in another concurrent investigational study or        intake of an investigational drug within 3 months or intake of        an investigational drug within a period of 5 half lives of that        drug prior to the screening visit    -   19. Blood loss in excess of 200 mL within 30 days of Day-1 in        excess of 500 mL within 90 days of Day-1 or in excess of 1350 mL        within 1 year of Day-1 or donation of blood products within 14        days of Day−1    -   20. Patient has a history of neoplastic disease ≦5 years prior        to signing informed consent, except for adequately treated basal        cell or squamous cell skin cancer or in situ cervical cancer.    -   21. Patient is pregnant (positive serum pregnancy test at        anytime during study participation) or breast-feeding, or is a        female expecting to conceive within the projected duration of        the study    -   22. Involvement in the planning and/or conduct of the study        (applies to both AstraZeneca staff and/or staff at the study        site)    -   23. Inability to understand or cooperate with the requirements        of the study    -   24. Patient is legally or mentally incapacitated    -   25. Patient has abnormal screening laboratory values as per the        guidelines listed below or other clinically significant,        unexplained laboratory abnormality according to the        investigator.        -   AST>1.5×upper limit of normal        -   ALT>1.5×upper limit of normal        -   Total bilirubin>1.5×upper limit of normal        -   Serum creatinine>2.0×upper limit of normal        -   DHEA and free testosterone upper limits    -   26. Patient has taken any of the following medications in the        time frame specified:    -    4 Weeks prior to screening and throughout the study period:        -   Potent and moderate CYP3A4 inhibitors, including but not            limited to: cyclosporine, systemic (oral/IV) itraconazole,            ketoconazole, fluconazole, erythromycin, clarithromycin,            telithromycin, nefazodone, HIV protease inhibitors,            aprepitant, verapamil, diltiazem        -   Potent and moderate CYP3A4 inducers, including but not            limited to: rifampicin, rifabutin, carbamazepine, phenytoin,            barbiturates, systemic glucocorticoids (replacements and            inhaled are permitted), nevirapine, efavirenz, pioglitazone,            primidone, St. John's wort        -   Potent and moderate CYP2C9 inhibitors, including but not            limited to amiodarone, fluconazole, miconazole, oxandralone:        -   Potent and moderate CYP2C9 inducers, including but not            limited to: carbamazepine, rifampin        -   Metformin    -    8 weeks. prior to screening and throughout the study period:        -   Oral contraception, Estrogen, Progestorone            Given the potential for a drug interaction between CYP3A4            substrates and NK3RA, the suitability of co-dosing and            maximum dose of CYP3A4 inhibitors should be guided by            approved label.

The use of concomitant medications that prolong the QT/QTc interval isprohibited. Slight modifications to the trial proposal were made as theprotocol evolved. For example in the original protocol the testosteroneinclusion criteria was to be above the upper limit of normal but waseventually revised to free testosterone at screening greater than 85% ofthe age-specific upper limit of normal (ULN).

The effect of NK3RA on testosterone and LH is being assessed.Specifically, 8 hours of intensive LH sampling (1 sample per 10 min) atbaseline and on day 7 and 28 was scheduled to capture changes of LH AUCand pulse frequency and amplitude over the dosing interval. LH pulseamplitude and frequency are mathematically modelled parameters from rawLH assay data. AUC is by definition a calculated entity influenced byfrequency and amplitude, hence it is possible to infer information aboutLH pulsatility from LH AUC.

Hourly measurement of FSH and free and total testosterone was performedduring these periods of intensive monitoring.

Plasma concentration-time profiles of NK3RA and its metabolite will beconstructed for each subject in the PK Analysis Population. For eachplasma concentration-time curve, the following PK parameters wasdetermined using noncompartmental methods with validated PK softwareCmax, time to maximum plasma concentration (Tmax), area under the curve(AUC0-8) for plasma concentration versus time curve.Clinical safety and tolerability, are being assessed by changes inphysical examinations, vital signs, body weight, clinical laboratorytests, adverse experiences, and electrocardiograms

Results

Interim data have been obtained from a total of 20 women with PolycysticOvary Syndrome (PCOS) who received treatment (NK3RA or placebo) in theongoing phase 2a study. This study is a 28 day, randomised, double blindplacebo controlled study with 4 treatment arms, NK3RA 20 mg once daily,20 mg twice daily, 40 mg twice daily and placebo in women with PCOS.

Tables 1 to 3 below summarise the number of patients with datasupportive, consistent and not supportive by treatment group (placeboand NK3RA).

In Table 1 the criteria used to determine what is supportive, consistentor not supportive are based on a qualitative evaluation of the changesin:

-   -   (a) LH area under the curve (AUC) between 0 and 8 hours AUC₍₀₋₈₎    -   (b) LH pulsatility parameters derived through deconvolution        (Veldhuis J D, Keenan D M, and Pincus S M. Motivations and        methods for analyzing pulsatile hormone secretion. Endocrine        Reviews. 2008, 29(7):823-864; and Liu P Y, Keenan D M, Kok P,        Padmanabhan V, O'Byrne K T, and Veldhuis J D. Sensitivity and        specificity of pulse detection using a new deconvolution method.        Am J Physiol Endocrinol Metab. 2009; 297(2):538-44)        -   i) average mass per LH pulse in 8 hours        -   ii) total number of LH pulses in 8 hours.

For LH, there is a relationship between the LH AUC₍₀₋₈₎ and LHpulsatility. In particular, reductions in LH AUC₍₀₋₈₎ that would beconsidered to be disease modifying in PCOS will manifest themselves inpulsatility parameters of average mass per LH pulse in 8 hours and/ortotal number of LH pulses in 8 hours. Mass per pulse (MPP) is definedthe amount of LH released per reconstructed LH pulse. The LH AUC₍₀₋₈₎can be more reliably measured compared to the pulsatility parameters andtherefore why both AUC₍₀₋₈₎ and pulsatility are considered ininterpreting the level of LH reduction.

LH criteria for Table 1 Not supportive Compared to baseline, LH AUCincreases on either day 7 or 28 AND both MPP and number of pulsesincrease on either day 7 or 28 Consistent Compared to baseline, eitherLH AUC decreases on both days 7 and 28 OR either MPP or number of pulsesdecrease on both days 7 and 28 Supportive Compared to baseline, both LHAUC decreases on both days 7 and 28 AND either MPP or number of pulsesdecrease on both days 7 and 28 Not evaluable Insufficient data availableto make a decision

In Table 2 the criteria used to determine what is supportive, consistentor not supportive are based on a qualitative evaluation of the changesin average (on an individual patient basis) free testosterone.

In Table 3 the criteria used to determine what is supportive, consistentor not supportive are based on a qualitative evaluation of the changesin average (on an individual patient basis) total testosterone.

Free T/Total T (evaluated independently) for Tables 2 and 3 Notsupportive Compared to baseline, there is an increase in testosterone onboth days 7 and 28 Consistent Compared to baseline, there is an decreasein testosterone on either days 7 and 28 Supportive Compared to baseline,there is an decrease in testosterone on both days 7 and 28 Not evaluableInsufficient data available to make a decision e.g. baseline is missing

Expert judgement was necessary in the application of these criteria andtherefore plots were evaluated independently by 3 AZ physicians. Themost common result across the 3 physicians determined provided theoverall evaluation for a single patient. These criteria were agreedbetween the 3 reviewers ahead of the plots being available for review.

TABLE 1 No. of patients LH Supportive Consistent Non-supportive NK3RA 23 3 Placebo 0 1 1 Total 2 4 4

TABLE 2 Free No. of patients testosterone Supportive ConsistentNon-supportive NK3RA 2 4 3 Placebo 0 1 1 Total 2 5 4

TABLE 3 Total No. of patients Testosterone Supportive ConsistentNon-supportive NK3RA 3 3 4 Placebo 0 0 3 Total 3 3 7

Only the number of patients with data that were evaluable for therelevant parameter is recorded in the tables. Some data were notevaluable, because the pre dose baseline was missed or samples weremissing or haemolysed during the 8 hour profile.

Example 1

3-(Methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide having the following structuremay be prepared as follows:

To 3-[(methylsulfonyl)amino]-2-phenylquinoline-4-carboxylic acid (1c)(20.00 g, 58.4 mmol) in ethyl acetate (180 ml) 1,1′-carbonyldiimidazole(13.26 g, 81.8 mmol) is added. The resulting slurry is heated to 50° C.and the reaction mixture stirred at 50° C. for 6 h.(S)-(−)-1-phenylpropylamine (11.85 g, 87.6 mmol) in ethyl acetate (15ml) is then added and the reaction mixture further heated to 70° C. andstirred for 8 h. The solution is then cooled to room temperature and theresidue partitioned between ethyl acetate and aqueous hydrochloric acid.The organic phase is then co-distilled with isopropanol to result in anisopropanol solution. This is then seeded and cooled. The solid iscollected by filtration, washed with isopropanol and dried to yield thetitle compound (19.74 g, 74%) as a solid. ¹H NMR (300 MHz, CDCl₃) δ 0.94(t, 3H), 1.97 (m, 2H), 3.44 (s, 3H), 5.17 (q, 1H), 5.47 (m, 2H), 7.32(d, 2H), 7.34 (d, 2H), 7.39 (m, 1H), 7.78 (m, 2H), 7.84 (m, 2H), 8.08(m, 1H), 8.30 (m, 2H), 8.42 (m, 2H). MS APCI, m/z=460 (M+1).

The starting acid,3-[(methylsulfonyl)amino]-2-phenylquinoline-4-carboxylic acid (1c), maybe prepared in the following manner:

N-(2-oxo-2-phenylethyl)methanesulfonamide (1 b)

A solution of 2-amino-1-phenylethanonehydrochloride (200 g, 1.165 mol)in NMP (800 mL) is formed and after cooling, methylsulfonyl chloride(117.3 mL, 1.515 mol) is added slowly. This is followed by the slowaddition of N-methylmorpholine (450.1 ml, 4.078 mol), the reactionmixture is then stirred at 0° C. for 1 h. The mixture is warmed, withbrine and seed then being added. The result slurry is cooled and solidcollected by filtration, washed with water and dried to yield the titlecompound (209.9 g, 85%) MS APCI, m/z=214 (M+1).

3-[(methylsulfonyl)amino]-2-phenylquinoline-4-carboxylic acid (1c)

A slurry of N-(2-oxo-2-phenylethyl)methanesulfonamide (1b) (100 g, 0.469mol) and isatin (69 g, 0.469 mol) in isopropanol (700 ml) is heated to50° C. Aqueous sodium hydroxide (133 ml, 2.334 mol) is added. Thereaction mixture is heated to 75° C. and stirred for 1 h. The resultingsolution is cooled and acidified with hydrochloric acid to pH4. Thesolution is seeded and further hydrochloric acid added to achieve pH2-3.The resulting solid is collected by filtration, washed with isopropanoland water and dried to yield the title compound (112.1 g, 65%). ¹H NMR(300 MHz, CDCl₃) δ 3.11 (s, 3H), 7.05 (d, 1H), 7.39 (d, 2H), 7.64 (m,2H), 7.78 (m, 1H), 8.06 (m, 1H), 8.19 (m, 1H), 8.47 (m, 1H), 10.03 (b,2H). MS APCI, m/z=343 (M+1).

Example 2

Method:

Population pharmacokinetic/pharmacodynamic (PK/PD) modeling of NK3RA andtestosterone concentrations from four phase I studies and one phase IIstudy was performed to quantify the exposure-response relationshipsbetween plasma concentrations of NK3RA and testosterone. PKPD analyseswere conducted using sequential approach via nonlinear mixed-effectsmodeling with NONMEM® VII. The PK model was developed first and thepredicted concentrations from the empirical Bayes estimates of the PKparameters were used in the PD response model building. The developedPKPD model was used to explore different dosing regimens [40 mg bi-daily(BID) vs 80 mg once-daily (QD)] targeting reduction of plasmatestosterone levels and predict PD response.

NONMEM is a software package, just like Microsoft Office. It is aspecialized software for the analysis of pharmacokinetic andpharmacodynamic data. NONMEM is an abbreviation of the full name“NONlinear Mixed-Effect Modeling” which was developed at the Universityof California at San Francisco by two professors, Lewis Sheiner andStuart Beal in the late 1970s and has become the “gold standard”, bothin the pharmaceutical industry and academia.

The NONMEM software is a regression program that specializes innon-linear systems. The population PK analysis was based on multipleregression using non-linear mixed effect models. Mixed effect modelsdescribe the influence of both fixed effects and random effects on adependent variable, e.g. plasma drug concentration or a clinicalendpoint. Fixed effects, THETA (θ) in NONMEM notation, are factors thatare either measured or controlled, e.g. CL and V. Random effects includeresidual error (ERR), epsilon (ε) in NONMEM notation, and betweensubject random effects, ETA (η) in NONMEM notation. Population PK mixedeffects models typically include four basic components: the structuralPK model, which predicts the plasma concentration as a function of timeand dose; the covariate model component, which describes the influenceof fixed effects (e.g. demography) on PK model population parameters;the between-subject variance component, which describes theinter-individual variation in PK parameters (after “correction” forfixed effects); and the residual error model components, which describesthe underlying distribution of the error in the measured PK variable.

Results:

Data including 3597 NK3RA PK observations and 786 testosteroneconcentrations from 139 healthy volunteers were investigated. Atwo-compartment model with first-order elimination best described NK3RAPK. Circadian rhythm of baseline testosterone concentrations was welldescribed by a cosine function. Indirect response model (inhibition ontestosterone production) was used to link the drug effect to PDresponse. The scheme of PKPD model is illustrated in FIG. 1. It wasconcluded that following 40 mg BID treatment, trough NK3RA concentrationwill be higher compared to 80 mg BID. The time above IC50 fortestosterone concentration after 40 mg BID of NK3RA is 80.9% time of thedosing interval compared to only 55.7% after 80 mg QD (FIG. 2). The meanpredicted peak testosterone concentration at steady state are lower andoverall less variable during 24 hrs for 40 mg BID dose compared to 80 mgQD dose (FIG. 3). These findings surprisingly suggest 40 mg BID dosingprovided better sustained testosterone suppression effect during doseinterval than 80 mg QD.

Conclusions:

Population pharmacokinetic and pharmacodynamic analysis demonstratesthat 40 mg administered twice a day is better than 80 mg once a day tomaximally suppress testosterone during the entire dosing interval.

FIGURES

FIG. 1 is the PKPD model scheme of NK3RA.

FIG. 2 is the predicted steady state NK3RA concentration afteradministering 40 mg BID or 80 mg QD of NK3RA.

FIG. 3 is the predicted steady state testosterone concentration afteradministering 40 mg BID or 80 mg QD of NK3RA.

1-10. (canceled)
 11. A method of modulating LH pulsatility in a patientin need thereof, comprising administering to said patient atherapeutically effective amount of3-(methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide (NK3RA):

or a pharmaceutically acceptable salt thereof.
 12. A method of treatingpolycystic ovarian syndrome in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of3-(methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide(NK3RA):

or a pharmaceutically acceptable salt thereof.
 13. The method accordingto claim 12 wherein the method is a method of treating amenorrhea,oligomenorrhea, or anovulation in polycystic ovarian syndrome.
 14. Themethod according to claim 11 wherein the method is a method ofrestorating fertility.
 15. (canceled)
 16. A method of treating PCOScomprising the step of determining whether the testosterone level of abiological sample taken from a patient is higher than the normal leveland, if it is, treating said patient with a therapeutically effectiveamount of3-(methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide(NK3RA):

or a pharmaceutically acceptable salt thereof.
 17. A method according toany one of claims 11-14 or 16 wherein3-(methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide(NK3RA) is administered in the form of a pharmaceutical compositioncomprising:3-(methanesulfonamido)-2-phenyl-N-[(1S)-1-phenylpropyl]quinoline-4-carboxamide(NK3RA); mannitol and microcrystalline cellulose; croscarmellose sodium,hydroxypropyl cellulose, sodium lauryl sulphate, and magnesium stearate.